This invention describes cloning and characterization of the canine IgE gene. The canine IgE gene was isolated using a human IgA constant region probe to clone a piece of the dog genome. IgA-containing cloned fragments of the dog genome were searched for IgE-related sequences. The identified areas were characterized in detail by nucleotide sequence analysis. This invention provides specific sequence information which permits targeted modulation of IgE-mediated immune responses.
The invention relates to DNA molecules encoding a canine IgE and species specific regions of canine IgE constant region. The invention comprises the DNA molecules, proteins encoded by the DNA molecules, antibodies to the proteins, cells transformed by the DNA molecules, assays employing the transformed cells, compounds identified by the assays and kits containing the DNA molecules or derivatives thereof.
Traditionally, hypersensitivity responses in the dog have been controlled by corticosteroid therapy which has adverse metabolic effects and produces generalized immunosuppression. The cloning and sequence determination of the canine IgE gene permits novel approaches to the control of IgE-mediated hypersensitivity reactions by facilitating targeting of the IgE molecule and its interaction with the IgE receptor. These approaches include, but are not limited to eliciting an immune response directed at specific peptide epitopes present in canine IgE to control allergic reactions and using the canine IgE sequence as pan of a screen to identify small molecules that alter IgE mediated responses to allergens.
Immunoglobulin (Ig) proteins consist of two identical light (L) chains and two identical heavy (H) chains. Both Ig L and H chains contain an amino-terminal variable region of approximately 110 amino acids that forms the antigen binding domain. The carboxy terminal constant (C) region domains of each chain is defined by two isotypes of IgL chain (kappa and lambda) and multiple isotypes of IgH chains ( mu, delta, gamma, epsilon and alpha which define IgM, IgD, IgG, IgE, and IgA, respectively). The IgH chain C regions contain the effector functions common to antibodies of a given isotype.
IgE antibodies are responsible for mediating allergic responses. IgE binds to mast cells through an Fce receptor and, when cross-linked by binding antigen, triggers a cascade of events-that leads to the release of allergic mediators. Because of the central role that IgE plays in mediating allergic reactions, the region of the Ige constant region involved in Fc.epsilon. receptor binding is of great interest. Inhibition of binding of IgE to its receptor on mast cells may be a way to control allergic responses.
Interestingly, of all five isotypes of immunoglobulin, the sequence of the Ig.epsilon. C region is the least well conserved across species. Consequently, studies of allergic reactions in a specific species are aided by having the primary amino acid sequence available for the Ig.epsilon. C region gene of that species.
The IgE antibody class plays a central role in type I immediate hypersensitivity. IgE binds to specific high-affinity receptors on mast cells and basophils and, when associated with antigen, triggers degranulation of vasoactive substances to produce allergic reactions. Because of its role in allergy, substantial effort has been made to understand how the Ig.epsilon. C region (which defines IgE) interacts with the Fc.alpha. receptor on mast cells and basophils to trigger degranulation upon binding antigen. These studies indicate that binding to the Fc.epsilon. receptor reside in the Ig.epsilon. CH3 and CH4 domains. Additional studies have used linear peptides to map the Ig.epsilon. binding site. In one of these studies, an octapeptide from the human Ig.epsilon. gene (Pro345-Phe-Asp-Leu-Phe-Ile-Arg-Lys352) inhibited passive sensitization, presumably by occupying the Fc.epsilon. receptor sites on cells (Nio et. al. 1993). The equivalent region of the canine Ig.epsilon. chain shares only 50% identity with this octapeptide (Canine sequence: Pro-Leu-Asp-Leu-Tyr-Val-His-Lys). Based on this observation, attempts to use IgE peptides involved in Fc.epsilon. receptor binding to modulate allergic reactions in dogs would require the use of peptides derived from the canine Ig.epsilon. sequence.
The sequences of the IgE constant regions from several species including human, rat and mouse have been reported. Peptides derived from known IgE sequences have been used to generate antibodies which alter IgE function. U.S. Pat. No. 5,091,313 is directed to the prevention or control of IgE-mediated allergic symptoms through the use of monoclonal or polyclonal antibodies raised against epitopes present in B cell-associated or soluble human IgE. WO90/15878 discloses the use of peptides derived from human, rat or mouse IgE sequences to generate antibodies which inhibit IgE-mediated mast cell degranulation. U.S. Pat. No. 4,223,016 discloses the use of peptides derived from IgE sequences for allergic desensitization.
The present invention identifies a species-specific sequence is of the canine IgE constant region. For therapeutic purposes, it may be desirable to generate antibodies against the IgE of the target species in order to maximize the affinity of the anti-IgE antibodies. In addition, screening assays aimed at the identification of small molecules which alter IgE mediated responses in the dog can be optimized through the use of canine IgE, the actual target.
Prior to the described invention, it was virtually impossible to design peptides which could be used to produce antibodies of specifically targeted against canine IgE. When IgE sequences from other species are used for this purpose, the resulting antibodies have reduced affinity for the canine IgE and, therefore, reduced efficacy compared with antibodies generated using the described invention. Further, the availability of the cloned canine IgE gene enables large quantities of the canine IgE protein to be produced recombinantly for use in drug development (e.g., small molecule screening, assay development and anti-IgE antibody generation).
The DNA of the present invention may be used to identify regions of the canine IgE which are homologous to those targeted in other species and to predict novel therapeutic targets. Therapeutically interesting portions of the sequence mad be expressed in chimeric proteins or used to produce peptides. These molecules or conjugate derivatives thereof may then be used, with or without adjuvants, as canine vaccines to treat or prevent IgE mediated-hypersensitivity responses. Alternately, the derived peptides or proteins may be used to produce monoclonal or polyclonal antibodies for passive treatment of IgE-mediated hypersensitivity.
The invention also provides a renewable source of canine IgE protein through its expression using recombinant DNA techniques. This provides material for establishing assays to monitor IgE-mediated immune responses as well as for developing screens to identify small molecules capable of disrupting IgE-mediated allergic reactions in the dog.